Sickle cell disease (SCD) is a debilitating genetic disorder that affects mainly African-Americans. It is characterized by red blood cell hemolysis, chronic anemia, and recurrent episodic pain. Elevated levels of endothelin-1 (ET-1), low red cell Mg2+ levels, and increased K+ efflux may all contribute to sickle erythrocytes (SRBC) abnormal physiology. However, therapeutic agents that target RBC membrane channels have not been developed, primarily because the cellular and molecular mechanisms that mediate these events have not been sufficiently described. Erythrocyte dehydration, a hallmark in SCD, is in part mediated by activation of the K/Cl cotransporter, Gardos channel, and Na+/Mg2+ exchanger transporters whose activities are elevated in SRBC. We demonstrated that the Gardos channel in human SRBC is coupled to ET-1 receptors and that ET-1 induces dehydration in vitro; the mouse SRBC Gardos channel is also coupled to ET-1 receptor and other cytokine receptors. Our preliminary data indicate that sickle transgenic mice treated with ET-1 receptor antagonists have reduce SRBC cellular dehydration. Furthermore, we present evidence that the Na+/Mg2+ exchanger activity is regulated by ET-1 in normal erythrocytes but not SRBC. Moreover, we demonstrated that ET-1 regulates the activity of casein kinase II (CK2), a ubiquitous serine/threonine protein kinase found in eukaryotic cells that phosphorylate acidic proteins. CK2 has been shown to participate in the maintenance of cell viability by regulating numerous cellular processes and functions, including metabolism, signal transduction, and transcription. The inhibition of CK2 blocked K/Cl cotransport and Gardos channel activity in vitro and attenuated deoxygenation-stimulated and ET-1-induced erythrocyte dehydration. Based on our preliminary data we will identify signaling pathways involved in ET-1-induced cellular cation metabolism and the role of CK2 in SRBC dehydration, thus integrating various aspects of SCD pathophysiology. The central hypothesis for the proposed research is that elevated levels of ET-1 stimulates K+ and Mg2+ flux via CK2 activity, which leads to SRBC dehydration, sickling and vascular occlusion. Our specific aims are: Aim I. Identify the role of casein kinase II on the ET-1- and hypoxia-stimulated K+ flux from sickle erythrocytes. Aim II. Identify the signaling pathways that modulate cellular Mg2+ levels upon ET-1 receptor activation in sickle erythrocytes. Aim III. Characterize the effects of ETA/ETB receptor antagonist treatment in a transgenic mouse model of sickle cell disease. The purpose of this research project is to elucidate which signaling molecules regulate ET-1-mediated erythrocyte dehydration in vivo and to develop novel targeted pharmacological approaches to ameliorate painful crises and vascular damage caused by entrapment of dehydrated irreversibly sickled erythrocytes. Our long-term goal is to develop efficacious interventions for the management of vascular complications in sickle cell disease. PUBLIC HEALTH RELEVANCE: Sickle cell disease (SCD) is one of the most prevalent genetic blood disorders and a major health problem in the United States (US) affecting nearly 80,000 persons and millions in other parts of the world, especially on the continent of Africa. These statistics underscore the importance of research in this area. The disease is characterized by chronic anemia due to hemoglobin S polymerization and red cell hemolysis and recurrent painful episodes. The pathophysiology in SCD is aggravated by elevated levels of endothelin-1 (ET-1), low Mg2+, and increased K+ efflux. Thus far, hydroxyurea has been the only drug approved for SCD therapy; it induces the production of fetal hemoglobin, which inhibits hemoglobin S polymerization and improves signs of subacute inflammation, however, not all patients benefit from this treatment. Therefore, a search for novel therapeutic strategies as proposed in this grant application is of the utmost importance. Our preliminary data show that ET-1 receptor antagonist block sickle erythrocyte dehydration in vivo and in vitro and as such, may represent a novel class of therapeutics for SCD. We plan to characterize mechanisms of ET-1-stimulated sickle red cell dehydration as a means to develop ET-1 receptor antagonists as a novel therapy in the treatment of sickle cell disease. [unreadable] [unreadable] [unreadable]